St13 protects against disordered acinar cell arachidonic acid pathway in chronic pancreatitis.

BACKGROUND: Early diagnosis and treatment of chronic pancreatitis (CP) are limited. In this study, St13, a co-chaperone protein, was investigated whether it constituted a novel regulatory target in CP. Meanwhile, we evaluated the value of micro-PET/CT in the early diagnosis of CP. METHODS: Data from healthy control individuals and patients with alcoholic CP (ACP) or non-ACP (nACP) were analysed. PRSS1 transgenic mice (PRSS1Tg) were treated with ethanol or caerulein to mimic the development of ACP or nACP, respectively. Pancreatic lipid metabolite profiling was performed in human and PRSS1Tg model mice. The potential functions of St13 were investigated by crossing PRSS1Tg mice with St13-/- mice via immunoprecipitation and lipid metabolomics. Micro-PET/CT was performed to evaluate pancreatic morphology and fibrosis in CP model. RESULTS: The arachidonic acid (AA) pathway ranked the most commonly dysregulated lipid pathway in ACP and nACP in human and mice. Knockout of St13 exacerbated fatty replacement and fibrosis in CP model. Sdf2l1 was identified as a binding partner of St13 as it stabilizes the IRE1α-XBP1s signalling pathway, which regulates COX-2, an important component in AA metabolism. Micro-PET/CT with 68Ga-FAPI-04 was useful for evaluating pancreatic morphology and fibrosis in CP model mice 2 weeks after modelling. CONCLUSION: St13 is functionally activated in acinar cells and protects against the cellular characteristics of CP by binding Sdf2l1, regulating AA pathway. 68Ga-FAPI-04 PET/CT may be a very valuable approach for the early diagnosis of CP. These findings thus provide novel insights into both diagnosis and treatment of CP.

Danshensu alleviates pseudo-typed SARS-CoV-2 induced mouse acute lung inflammation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can induce acute inflammatory response like acute lung inflammation (ALI) or acute respiratory distress syndrome, leading to severe progression and mortality. Therapeutics for treatment of SARS-CoV-2-triggered respiratory inflammation are urgent to be discovered. Our previous study shows that Salvianolic acid C potently inhibits SARS-CoV-2 infection. In this study, we investigated the antiviral effects of a Salvia miltiorrhiza compound, Danshensu, in vitro and in vivo, including the mechanism of S protein-mediated virus attachment and entry into target cells. In authentic and pseudo-typed virus assays in vitro, Danshensu displayed a potent antiviral activity against SARS-CoV-2 with EC50 of 0.97 µM, and potently inhibited the entry of SARS-CoV-2 S protein-pseudo-typed virus (SARS-CoV-2 S) into ACE2-overexpressed HEK-293T cells (IC50 = 0.31 µM) and Vero-E6 cell (IC50 = 4.97 µM). Mice received SARS-CoV-2 S via trachea to induce ALI, while the VSV-G treated mice served as controls. The mice were administered Danshensu (25, 50, 100 mg/kg, i.v., once) or Danshensu (25, 50, 100 mg·kg-1·d-1, oral administration, for 7 days) before SARS-CoV-2 S infection. We showed that SARS-CoV-2 S infection induced severe inflammatory cell infiltration, severely damaged lung tissue structure, highly expressed levels of inflammatory cytokines, and activated TLR4 and hyperphosphorylation of the NF-κB p65; the high expression of angiotensinogen (AGT) and low expression of ACE2 at the mRNA level in the lung tissue were also observed. Both oral and intravenous pretreatment with Danshensu dose-dependently alleviated the pathological alterations in mice infected with SARS-CoV-2 S. This study not only establishes a mouse model of pseudo-typed SARS-CoV-2 (SARS-CoV-2 S) induced ALI, but also demonstrates that Danshensu is a potential treatment for COVID-19 patients to inhibit the lung inflammatory response.

Long noncoding RNA CCAT2 functions as a competitive endogenous RNA to regulate FOXC1 expression by sponging miR-23b-5p in lung adenocarcinoma.

Long noncoding RNA (lncRNA) may regulate the process of tumor formation. Although lncRNA CCAT2 has been identified as a key point in many diseases, its pathophysiological mechanism in lung adenocarcinoma remains unknown. We measured the expression level of CCAT2 in lung adenocarcinoma cells and normal lung epithelial cell line BEAS-2B by quantitative real-time polymerase chain reaction (qRT-PCR). As well, cell migration and proliferation were detected by transwell detection and CCK8 assay. At the same time, the new target point of CCAT2 was confirmed with bioinformatics analysis and dual-luciferase reporter assay. In addition, potential mechanisms were studied by Western blot analysis and RNA immunoprecipitation (RIP) analysis. The expression of CCAT2 was upregulated obviously in lung adenocarcinoma cells. Cell function analysis showed that upregulation of CCAT2 significantly promoted cell proliferation and migration, and reduction of CCAT2 inhibited cell migration and proliferation. In addition, CCAT2 positively regulated the expression of FOXC1 by competitive binding with miR-23b-5p. These findings indicated that CCAT2 may act as a competitive endogenous RNA (ceRNA) to regulate FOXC1 expression by competitively binding miR-23b-5p in lung adenocarcinoma.

Synthesis and biological evaluation of substituted 1,2,3-benzotriazines and pyrido[3,2-d]-1,2,3-triazines as inhibitors of vascular endothelial growth factor receptor-2.

A novel series of substituted 1,2,3-benzotriazines and pyrido[3,2-d]-1,2,3-triazines were synthesized. The abilities of these compounds to inhibit the VEGFR-2 kinase activity and the proliferation of human microvascular endothelial cells (MVECs) were determined. 6-Methoxy-4-substituted-1,2,3-benzotriazines and 4-substituted-6-chloro-pyrido[3,2-d]-1,2,3-triazines have the abilities of inhibiting the VEGFR-2 kinase activity, but only the 4-substituted-6-chloro-pyrido[3,2-d]-1,2,3-triazines exhibit good growth inhibitory effects on MVECs. Compound 6-chloro-4-(3-trifluoromethylanilino)-pyrido[3,2-d][1,2,3]triazin (11d) is less half active than PTK787 to inhibit the VEGFR-2 kinase activity, but is more active than PTK787 to inhibit the growth of MVECs. The potential binding modes of 6d, 11d, and CTZ12 in complex with their putative intracellular target, VEGFR-2, were predicted using Surflex-Dock.

[Application of expansion bilateral delto-pectoral thin skin flap in the repair of extensive scar in faciocervical region].

OBJECTIVE: To contrive an effective method of repairing the scar in bilateral faciocervical region. METHODS: Between April 2009 and February 2012, 9 patients with large scars on face and neck due to burn and scald were treated. There were 5 cases with face scars and 4 cervical scars. Their average age was 33 years (range: 23-48 years). The disease duration was 6 months to 20 years (mean: 6.5 years). The scar area was 12 cm × 7 cm to 22 cm × 26 cm. The soft tissue expanders (600-800 ml in volume) were implanted in delto-pectoral zone in one-stage operation. In two-stage operation, after the resection of cervical scars, the defects were repaired with delto-pectoral perforator flaps. In 5 facial scar cases, skin flap pedicle division was performed at Week 3. After the resection of scars, all wounds were repaired by expansion flap. The donor sites were sutured directly. The area of removed scar and the status of flap blood supply were observed. And the texture of flaps and patient satisfaction score were followed up for 6-30 months. RESULTS: Mild congestion of flap occurred postoperatively 1 case. The other flaps survived successfully. The flaps of 2 cases appeared bulky after transposition and flap repair was performed at Month 6. The appearance, texture, and color of flaps were similar to those at the donor sites. And there was an excellent match of flaps and recipient place.The patient satisfaction score was 7.6 ± 2.3. All achieved satisfactory functional and aesthetic outcomes. CONCLUSION: The method has many advantages and its clinical application is both safe and effective.

[Clinical efficacy of keloids treated by surgical ablation and immediate postoperative adjuvant radiotherapy].

OBJECTIVE: To evaluate the clinical efficacy of intralesional excision and immediate postoperative adjuvant radiotherapy in the treatment of keloids. METHODS: Eighty-one patients with a combined total of 86 keloids were treated with 6 MeV electron beam radiotherapy after surgical intralesional excision of keloids. All received a total dose of 15 - 20 Gy for 5 consecutive days beginning the day of surgery. The time interval between keloid excision and the delivery of first fraction of radiotherapy was < 6 h. The post-operative follow-up period was 12 - 31 months. RESULTS: Forty-three cases yielded excellent outcomes and there were 18 fair cases. The overall effective rate was 85.9%. There were 10 recurrent cases. Only adverse effects such as delayed wound-healing and telangiectasias (11.3%) were found. Neither severe complications nor secondary malignancies occurred. CONCLUSION: Intralesional excision of keloid and postoperative electron radiotherapy are well-tolerated and efficacious in the prevention of keloid recurrence.

Ethyl 2-[(Z)-2-benzyl-hydrazin-1-yl-idene]-2-bromo-acetate.

In the title compound, C(11)H(13)BrN(2)O(2), the dihedral angle between the phenyl ring and the almost planar (r.m.s. deviation = 0.011 Å) C-C(Br)=N-N(H)- fragment is 74.94 (16)°. In the crystal, mol-ecules are linked by N-H⋯O hydrogen bonds, which generate C(6) chains propagating in [010]. Weak aromatic π-π stacking [centroid-centroid separation = 3.784 (3) Å] may also help to consolidate the packing.

ATF6 aggravates acinar cell apoptosis and injury by regulating p53/AIFM2 transcription in Severe Acute Pancreatitis.

Background: There is no curative therapy for severe acute pancreatitis (SAP) due to poor understanding of its molecular mechanisms. Endoplasmic reticulum (ER) stress is involved in SAP and increased expression of ATF6 has been detected in SAP patients. Here, we aimed to investigate the role of ATF6 in a preclinical SAP mouse model and characterize its regulatory mechanism. Methods: Pancreatic tissues of healthy and SAP patients were collected during surgery. Humanized PRSS1 transgenic mice were treated with caerulein to mimic the SAP development, which was crossed to an ATF6 knockout mouse line, and pancreatic tissues from the resulting pups were screened by proteomics. Adenovirus-mediated delivery to the pancreas of SAP mice was used for shRNA-based knockdown or overexpression. The potential functions and mechanisms of ATF6 were clarified by immunofluorescence, immunoelectron microscopy, Western blotting, qRT-PCR, ChIP-qPCR and luciferase reporter assay. Results: Increased expression of ATF6 was associated with elevated apoptosis, ER and mitochondrial disorder in pancreatic tissues from SAP patients and PRSS1 mice. Knockout of ATF6 in SAP mice attenuated acinar injury, apoptosis and ER disorder. AIFM2, known as a p53 target gene, was identified as a downstream regulatory partner of ATF6, whose expression was increased in SAP. Functionally, AIFM2 could reestablish the pathological disorder in SAP tissues in the absence of ATF6. p53 expression was also increased in SAP mice, which was downregulated by ATF6 knockout. p53 knockout significantly suppressed acinar apoptosis and injury in SAP model. Mechanistically, ATF6 promoted AIFM2 transcription by binding to p53 and AIFM2 promoters. Conclusion: These results reveal that ATF6/p53/AIFM2 pathway plays a critical role in acinar apoptosis during SAP progression, highlighting novel therapeutic target molecules for SAP.

EMC6 regulates acinar apoptosis via APAF1 in acute and chronic pancreatitis.

Treatment of acute pancreatitis (AP) and chronic pancreatitis (CP) remains problematic due to a lack of knowledge about disease-specific regulatory targets and mechanisms. The purpose of this study was to screen proteins related to endoplasmic reticulum (ER) stress and apoptosis pathways that may play a role in pancreatitis. Human pancreatic tissues including AP, CP, and healthy volunteers were collected during surgery. Humanized PRSS1 (protease serine 1) transgenic (PRSS1Tg) mice were constructed and treated with caerulein to mimic the development of human AP and CP. Potential regulatory proteins in pancreatitis were identified by proteomic screen using pancreatic tissues of PRSS1Tg AP mice. Adenoviral shRNA-mediated knockdown of identified proteins, followed by functional assays was performed to validate their roles. Functional analyses included transmission electron microscopy for ultrastructural analysis; qRT-PCR, western blotting, co-immunoprecipitation, immunohistochemistry, and immunofluorescence for assessment of gene or protein expression, and TUNEL assays for assessment of acinar cell apoptosis. Humanized PRSS1Tg mice could mimic the development of human pancreatic inflammatory diseases. EMC6 and APAF1 were identified as potential regulatory molecules in AP and CP models by proteomic analysis. Both EMC6 and APAF1 regulated apoptosis and inflammatory injury in pancreatic inflammatory diseases. Moreover, APAF1 was regulated by EMC6, induced apoptosis to injure acinar cells and promoted inflammation. In the progression of pancreatitis, EMC6 was activated and then upregulated APAF1 to induce acinar cell apoptosis and inflammatory injury. These findings suggest that EMC6 may be a new therapeutic target for the treatment of pancreatic inflammatory diseases.

Potential of bone marrow mesenchymal stem cells in rejuvenation of the aged skin of rats.

The aim of the present study was to evaluate the anti-aging effects of bone marrow-mesenchymal stem cells (BM-MSCs) in a D-galactose-induced skin aging rat model. Male Sprague Dawley rats were randomly divided into four groups (n=10/group) as follows: Normal control group; skin aging model group; MSC-treated group by subcutaneous multi-point injection. The skin aging model was established by a daily subcutaneous injection of 15% D-galactose (1,000 mg/kg) for 8 weeks. Rats in the MSC-treated groups were administered 3×106/ml BM-MSCs/green fluorescent protein (GFP) for 4 weeks, administered once per week. Oxidative/antioxidative parameters were evaluated, and morphological and ultrastructure analyses were performed. Rats in the model group exhibited the typical changes of aging skin. Compared with the control group, rats in the model group had significantly increased malondialdehyde (MDA) content (P<0.01), and decreased serum superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities (P<0.05). MSC treatment markedly ameliorated aging-induced oxidative stress in the skin. Histologically, rats in the model group exhibited loosely arranged epidermal cell layers and disorganized collagen fibers. BM-MSC treatment significantly improved the histological abnormalities, which was similar to those in the control group. In addition, 7 days after the final cell transplantation, GFP-positive cells were observed by fluorescence microscopy to be distributed in the dermis. Injection of BM-MSCs significantly improved the D-galactose-induced histological abnormalities of the skin, by promoting an antioxidant response and ameliorating oxidative stress in aged skin. Thus, BM-MSCs may be beneficial in the rejuvenation of aged skin.

Effects of serum of the rats ventilated with high tidal volume on endothelial cell permeability and therapeutic effects of ulinastatin.

BACKGROUND: With the widespread use of ventilators in treating critically ill patients, the morbidity of ventilator-induced lung injury (VILI) is increasing accordingly. VILI is characterized by a considerable increase in microvascular leakiness and activation of inflammatory processes. In this study we investigated the effects of inflammatory mediators in VILI rat serum on endothelial cytoskeleton and monolayer cellular permeability, as well as the therapeutic effect of ulinastatin, to explore the pathogenesis and the relationship between biotrauma and lung oedema induced by VILI. METHODS: Thirty healthy male Sprague-Dawley rats were randomly divided into three groups: group A (normal tidal volume ventilation), group B (high tidal volume ventilation) and group C (high tidal volume ventilation plus ulinastatin). The serum of each rat after ventilation was added to endothelial cell line ECV-304 medium for two hours to observe the effects of serum and/or ulinastatin on endothelial fibrous actin and permeability. RESULTS: Compared to rats ventilated with normal tidal volume, serum of rats ventilated with high tidal volume caused a striking reorganization of actin cytoskeleton with a weakening of fluorescent intensity at the peripheral filament bands and formation of the long and thick stress fibres in the centre resulting in endothelial contraction and higher permeability. Prior treatment with ulinastatin lessened the above changes significantly. The changes of permeability coefficient of endothelial permeability after group A, B or C rats serum stimulation were (6.95 +/- 1.66)%, (27.50 +/- 7.77)% and (17.71 +/- 4.66)% respectively with statistically significant differences (P < 0.05) among the three groups. CONCLUSIONS: The proinflammatory mediators in the serum of the rats given high tidal volume ventilation increases endothelial permeability by reorganizing actin cytoskeleton, and pretreatment with ulinastatin lessens the permeability by inhibiting of proinflammatory mediators.

[Transcatheter arterial chemoembolization in the treatment of hepatocellular carcinoma].

OBJECTIVE: To evaluate the effect of transcatheter arterial chemoembolization (TACE) on the result and the prognosis of hepatocellular carcinoma (HCC) at systemic, cellular, genetic and molecular levels. METHODS: Patients with histologically proven HCC were divided into two groups: 81 patients in Group A undergoing TACE before operation and 58 patients in Group B treated with surgical resection alone. The degree of apoptosis was analyzed by transferase -mediated dUTP nick end labeling (TUNEL) stain. The expressions of bcl-2, bax, p53, Ki-67 and PCNA proteins were detected by immunohistochemical method. The changes of these markers, tumor necrosis, encapsulation, volume, metastasis, recurrence and cumulative survival in each group were retrospectively analyzed. RESULTS: The more tumor necrosis, apoptosis, encapsulation and tumor shrinkage observed, and the less recurrence resulted from TACE in group A than in group B. The cumulative 1-, 2-, and 3-year survival rates and median survival time were 84.0%, 67.9%, 40.7%, and 803.3 days in group A patients; they were 72.4%, 55.2%, 24.1%, and 742.5 days in group B patients (P < 0.05). CONCLUSION: Preoperative transcatheter arterial chemoembolization is safe and effective as an auxiliary preparatory means before surgical treatment of hepatocellular carcinoma as it may improve the survival of HCC patients.

[Method for human peripheral blood eosinophil isolation for patch-clamp study].

OBJECTIVE: To establish a rapid and economic method for isolating human peripheral blood eosinophils with high viability for patch-clamp studies and investigate the electrophysiological properties of Ca(2+)-activated K(+) channel of the isolated cells. METHODS: Peripheral blood eosinophils were isolated by modified discontinuous Percoll density gradient centrifugation, and the electric currents in the single Ca(2+)-activated K(+) channels of the cells were recorded using patch-clamp technique with cell-attached configuration. RESULTS: The purity of the eosinophils from healthy donors reached (90.5+/-1.6)%, with a viability rate over 99% and recovery rate of (48.2+/-6.9)%. The isolated cells were morphologically intact, from which Ca(2+)-activated K(+) channel activity could be detected. CONCLUSION: The peripheral blood eosinophils isolated using this rapid, simple and highly efficient method are characterized by high purity and viability without obvious cellular injuries, which are ideal for patch-clamp studies.

[Effect of different tidal volume ventilation on rat bronchial and alveolar epithelial cell apoptosis].

OBJECTIVE: To investigate the changes in bronchial and alveolar epithelial cell apoptosis after mechanical ventilation with different tidal volumes and explore the mechanism and significance of cell apoptosis in ventilation-induced lung injury. METHODS: Thirty healthy male Sprague-Dawley rats were randomly divided into groups A, B and C to receive tracheotomy under anesthesia for ventilation with different tidal volumes (VT) for 4 h (VT of 10 ml/kg in group A, 20 ml/kg in group B and 40 ml/kg in group C). Lung histopathology was assessed after the ventilation and compared between the 3 groups. The severity of lung injury and bronchial epithelial edema was evaluated, and the extent and location of cell apoptosis in the bronchus and lung tissues were studied by transferase d-UTP end-labeling (TUNEL) assay and immunohistochemistry. RESULTS: After mechanical ventilation for 4 h, lung injury and bronchial epithelial edema increased with the increment of VT. By TUNEL assay, the apoptosis index of both alveolar and bronchial epithelial cells was found to increase with VT. Immunohistochemical study identified enhanced caspase-3 expression in the cytoplasm and nuclei of airway and alveolar epithelial cells and vascular endothelial cells in rats with high VT ventilation, and the enhancement was especially obvious in the bronchial epithelial cells. CONCLUSION: Mechanical ventilation with different VTs causes consistent changes in histopathology, cell apoptosis and caspase-3 expression in rats, which support the presumption that cell apoptosis plays an important role in the pathogenesis of high VT ventilation-induced lung injury.

Cloning and Sequence Analysis of the Chitinase Gene of Spodoptera litura Nuclear Polyhedrosis Virus.

Using AcMNPV chiA-containing fragment as a probe, the chitinase gene of Spodoptera litura nuclear polyhedrosis virus (SpltNPV) was localized in two contiguous fragments, XbaI 5.1 kb and 2.1 kb, and the intact SpltNPV chiA gene was obtained by cloning and sequencing those two fragments. The complete open reading frame of the gene was 1 695 nucleotide long, encoding a putative protein of 564 amino acids with molecular weight of 62.9 kD. Sequence analysis further revealed that the 5' noncoding region had a baculovirus late promoter motif TAAG. A polyadenylation signal, AATAAA, was located dowmnstream of the translation stop codon. A putative signal peptide was present at the N-terminus of the protein. This gene shares 57% homology with AcMNPV chitinase gene.

Relationship between apoptosis and invasive and metastatic potential of hepatocellular carcinoma.

OBJECTIVE: To study relationship between apoptosis and invasive and metastatic potential of hepatocellular carcinoma(HCC). METHODS: Apoptotic rate (AR), proliferative index (PI) and S-phase fraction (SPF) were measured by flow cytometry, and p170, p21 and nucleoside diphosphate kinase (ndpk) by strept avidin-biotin complex immunohistochemical technique in 57 patients with HCC. RESULTS: In this group, AR was 1.77%+/-0.19%, SPF 12.55%+/-0.68%, and PI 20.91%+/-1.12% (r=-0.173). p170, p21 and ndpk positive rates were 61.36%, 68.18%, 52.27% respectively in patients with a mean AR of 1.77% (all P<0.05). In patients with positive tumor invasiveness and metastasis, ndpk (+) was 43.75%, p21 (+) 75.00%, p170 (+) 65.63%, AR 1.12%+/-0.16%, PI 23.78%+/-1.48%, and SPF 13.90%+/-0.99%. In patients with negative invasiveness and metastasis, however, ndpk (+) was 80.00%, p21 (+) 44.00%, p170 (+) 36.00%, AR 2.32%+/-0.52%, PI 18.53%+/-0.82% and SPF 11.43%+/-0.70%. CONCLUSION: Apoptosis of HCC is negatively correlated with its invasive and metastatic potential or other factors as proliferative activity, p21, p170 and ndpk.

[The comparison between the vascular endothelial cells special cdc42-deficient heterozygous mice and the non-knockout mice on lung tissue pathological change and vasopermeability in acute lung injury].

OBJECTIVE: To compare the change of lung tissue and vasopermeability between the vascular endothelial cells special cdc42-deficient heterozygous mice and the non-knockout mice in acute lung injury. METHODS: The mice with vascular endothelial cell-specific expression of cre recombinase were crossed with cdc42(flox/flox) mice. The cdc42(flox/+)Cre(+/-) F1 offspring mice were crossed back with cdc42(flox/flox) mice, resulting in the F2 generation mice with three genotypes, namely cdc42(flox/+)Cre(+/-), cdc42(flox/flox)Cre(-/-) and cdc42(flox/+)Cre(+/-). The heterozygous mice with cdc42(flox/+)Cre(+/-) genotype were selected as the model mice, with the other two genotype groups as the control. After intratracheal instillation of 2 mg/kg LPS to induce acute lung injury, the mice were sacrificed to examine the lung pathologies, lung wet/dry ratio and lung microvascular permeability. RESULTS: The heterozygous mice with cdc42 gene knockout (cdc42(flox/+)Cre(+/-)) showed no significant differences from the two control groups in the lung pathological score, lung wet/dry ratio or the lung microvascular permeability coefficient. CONCLUSION: There were no significant difference on lung tissue and vasopermeability between the vascular endothelial cells special cdc42-deficient heterozygous mice and the non-knockout mice.

The generation of the endothelial specific cdc42-deficient mice and the effect of cdc42 deletion on the angiogenesis and embryonic development.

BACKGROUND: High microvascular permeability plays an essential role in pathological process of multiple diseases such as septic shock, acute lung injury and acute respiratory distress syndrome, and burns. Inhibiting hyperpermeability is significant for controlling these conditions. Cdc42, as a main member of the small Rho GTPase family, plays a critical role in controlling and regulating the endothelial junctional permeability. We aimed to generate and identify endothelial specific cdc42-deficient mice by the Cre/loxp recombination approach, for examination in an animal model of the contribution of the cdc42 gene in the microvascular barrier function. METHODS: We crossed cdc42(Flox/Flox) mice with mice expressing endothelial cell-specific Cre recombinase, and the offspring with the genotype cdc42(Flox/+)Tie2Cre(+/-) were back-crossed with the cdc42(Flox/Flox) mice. The cdc42(Flox/Flox)Tie2Cre(+/-) mice in the F2 generation were the target mice. If the cdc42 deficient mice did not survive, we would observe the cdc42 deficient mice embryos, and compare them with wild-type mice embryos. RESULTS: Cdc42(flox/+)Cre(+/-) mice were mated with the cdc42(Flox/Flox) mice and among the living offspring there were no cdc42(Flox/Flox)Cre(+/-) target mice. We found the endothelial special cdc42 deficient embryos at the E7.5-E16.5 stage. We observed that cdc42 deficient embryos were much smaller, had fewer vessels and were a little more swollen compared with the wild-type embryos. CONCLUSIONS: Endothelial specific knockout of cdc42 caused embryonic lethality and the mice did not survive to birth. The target embryos were much smaller, had fewer vessels and were a little more swollen compared with the wild-type embryos. These results demonstrated that the cdc42 plays an important role in development of embryos and in development of microvessels as well as microvascular permeability.

Some insights into mechanism for binding and drug resistance of wild type and I50V V82A and I84V mutations in HIV-1 protease with GRL-98065 inhibitor from molecular dynamic simulations.

The single mutations I50V, V82A and I84V are considered as the key residue mutations of the HIV-1 protease drug resistance. The rank of calculated absolute binding free energies using MM-PBSA method is in excellent agreement with experimental result. Enthalpic and entropic balance is analyzed to explain resistance in I50V and V82A having a higher entropic contribution than in the wild type (WT) complex. The reduced van der Waals energy explains the drug resistance of I84V to GRL-98065. Detailed binding free energies between GRL-98065 and individual protein residues are calculated to provide insights into the inhibitor-protein binding and drug-resistant mechanism. Our results show I50V and V82A have larger structural changes than I84V compared with WT.

[Therapeutic effect of tiotropium bromide powder inhalation in patients with stable bronchiectasis].

OBJECTIVE: To observe the therapeutic effect of tiotropium bromide powder inhalation on stable bronchiectasis. METHODS: Twenty-two patients with stable bronchiectasis received inhalation of totropium bromide powder at the daily dose of 18 microg, and on days 1 and 28, the patients were examined for forced expiratory volume in one second (FEVl), predicted value [FEVl(%)], forced expiratory volume (FEV), and FEVl/FVC. The symptom score and BODE index were also recorded. RESULTS: After 1 month of inhalation therapy, the FEV1% of the patients showed a moderate increase but the increment was not statistically significant (t=-1.875, P>0.05); the symptom score and BODE index decreased significantly after the therapy (t=7.091, P<0.001; t=2.982, P<0.05). CONCLUSION: Long-term inhalation of tiotropium bromide powder can improve the clinical symptoms and BODE index and enhance the exercise tolerance and quality of life of the patients with bronchiectasis.